Quantitative alterations of S-100 protein and neuron specific enolase in the rat nervous system after chronic 2,5-hexanedione exposure

The regional changes in quantities of the glial S-100 protein and the neuron specific enolase in the rat nervous system have been studied after long-term exposure to 2,5-hexanedione. The wet weights of most of the examined nervous tissues were found to be reduced, with an extensive effect seen in the brain stem. Using dot immunobinding assays, the concentrations of S-100 were found to be increased in most of the examined tissues, but unaffected in the brain stem. The total amount of S-100 per tissue was markedly reduced in the brain stem. The content of neuron specific enolase was reduced only in the brain stem. Thus the effects of 2,5-hexanedione on the nervous system varied regionally. The brain stem was severely atrophied with a reduction of neuronal as well as of glial marker proteins. Other brain regions contained increased glial cell marker proteins as signs of progressive astroglial reactions.     

Isolation and purification of calcium-binding protein from electroplax of Electrophorus electricus.

An acidic calcium-binding phosphoprotein has been isolated from a cholinergic tissue, electroplax from Electrophorus electricus. The purification procedures included (NH4)2SO4 fractionation, boiling treatment, ECTEOLA-cellulose chromatography, and gel filtration on Sephadex G-100. Experiments were performed to compare this protein and a calcium-binding protein isolated from mammalian brain, adrenal medulla, and testis. These experiments showed that the two proteins were identical in terms of molecular weight (14 000), calcium-binding dissociation constant (kd=2.1-10(-5) M), electrophoretic mobility at pH 8.7 in 15% polyacrylamide gels, and phosphorus content (1 mol phosphorus per mol protein). In addition, the two proteins had similar amino acid compositions and peptide maps. Although the electroplax protein was not present in eel skeletal muscle, preliminary experiments indicated that small amounts of the protein were present in other eel tissues, namely brain, liver and spleen. These results suggest an identity between the electroplax and mammalian calcium-binding proteins and extend the findind of comparatively large amounts of the protein from mammalian nervous tissue to a cholinergic nervous tissue, electroplax. The close similarity of the proteins suggests a conservation of structure during evolution which may be required to fulfill a role in neuronal function.     

Comparison of acetylcholine and alpha-bungarotoxin binding sites in insects and vertebrates

1. The nervous tissue of locusts contains high affinity as well as low affinity binding sites for acetylcholine which display a similar nicotinic pharmacology. 2. Hill plot analysis indicated a non-cooperative binding of acetylcholine. 3. In membrane preparations from locust ganglia and mouse brain the number of binding sites for ACh was about ten fold lower than for BGTX, whereas in membranes from electric tissue both sites occurred in similar concentrations. 4. Drug binding studies suggest that the high affinity binding sites for ACh and BGTX in preparations from insect and mouse are different; whereas in electric tissue both sites are very similar. 5. Precipitation experiments using immobilized BGTX and specific antibodies indicated that in insect nervous tissue as in electric tissue the ACh and BGTX binding sites are located on the same receptor molecule and occupy distinct partially overlapping binding sites, whereas in the vertebrate brain both sites are located on distinct binding proteins.     

Immunohistochemical study of doublecortin and nucleostemin in canine brain

Finding a marker of neural stem cells remains a medical research priority. It was reported that the proteins doublecortin and nucleostemin were related with stem/progenitor cells in central nervous system. The aim of the present immunohistochemical study was to evaluate the expression of these proteins and their pattern of distribution in canine brain, including age-related changes, and in non-nervous tissues. We found that doublecortin had a more specific expression pattern, related with neurogenesis and neuronal migration, while nucleostemin was expressed in most cells of almost every tissue studied. The immunolabeling of both proteins decreased with age. We may conclude that nucleostemin is not a specific marker of stem/progenitor cells in the dog. Doublecortin, however, is not an exclusive marker of neural stem cells, but also of neuronal precursors.Key words: nucleostemin, doublecortin, stem cells, dog brain, aging.     

Modulation of brain protein phosphorylation by the S-100 protein

The effects of the nervous system specific protein, S-100, on protein phosphorylation in rat brain is examined. The S-100 protein inhibits the phosphorylation of several soluble brain proteins in a calcium dependent fashion. The most potent effect exhibited by S-100 was on the phosphorylation of a protein having a molecular weight of 73,000. The data suggest that the calcium binding S-100 protein, for which a function has not yet been assigned, may modulate calcium dependent phosphorylation of selected brain proteins.     

The reconstruction and analysis of tissue specific human metabolic networks

Human tissues have distinct biological functions. Many proteins/enzymes are known to be expressed only in specific tissues and therefore the metabolic networks in various tissues are different. Though high quality global human metabolic networks and metabolic networks for certain tissues such as liver have already been studied, a systematic study of tissue specific metabolic networks for all main tissues is still missing. In this work, we reconstruct the tissue specific metabolic networks for 15 main tissues in human based on the previously reconstructed Edinburgh Human Metabolic Network (EHMN). The tissue information is firstly obtained for enzymes from Human Protein Reference Database (HPRD) and UniprotKB databases and transfers to reactions through the enzyme-reaction relationships in EHMN. As our knowledge of tissue distribution of proteins is still very limited, we replenish the tissue information of the metabolic network based on network connectivity analysis and thorough examination of the literature. Finally, about 80% of proteins and reactions in EHMN are determined to be in at least one of the 15 tissues. To validate the quality of the tissue specific network, the brain specific metabolic network is taken as an example for functional module analysis and the results reveal that the function of the brain metabolic network is closely related with its function as the centre of the human nervous system. The tissue specific human metabolic networks are available at .     

Isolation and some physico-chemical properties of a new neurospecific protein 10-40-4 from human brain

A method for isolation of a neurospecific protein 10-40-4 from human brain has been elaborated. This procedure includes immunoaffinity chromatography of a Sepharose 4B-IgG fraction of rabbit antisera against the protein fraction containing the antigen. The isolated protein cannot be detected in protein extracts of various organs and human blood serum by immunochemical methods. This indicates that the protein is specific for nervous tissue. The values of molecular weight (74 000) and pI (4.7) of the isolated protein suggest that the protein does not contain the carbohydrate component and reveals limited tissue specificity. The properties of protein 10-40-4 differ from those of the well-known neurospecific proteins, such as S-100, enolase 14-3-2 and glial fibrillar acid protein GFA.     

Endothelin-3 is a novel neuropeptide: isolation and sequence determination of endothelin-1 and endothelin-3 in porcine brain

The molecular forms of endothelin (ET) related peptides were investigated in porcine brain by using high performance liquid chromatography coupled with three specific radioimmunoassays. ET-1 and its oxidized form were isolated and sequenced as in the case of porcine spinal cord. A very small amount of big ET-1 (1-39) and its C-terminal fragment (big ET-1 (22-39] were also detected. Furthermore, immunoreactive (ir)-ET-3 was isolated and sequenced; its partial primary structure was identical to that of human (rat) ET-3. The concentrations of ir-ET-1 and ir-ET-3 in porcine brain were 140 fmol/g tissue and 5 fmol/g tissue, respectively. These results indicate that besides ET-1, ET-3 is a novel neuropeptide in the central nervous system.     

Characteristics of brain gastrin/cholecystokinin-binding proteins

Gastrin/cholecystokinin-binding proteins were purified using the column affinity chromatography on immobilized pig tetragastrin and cholecystokinin. Immunoblotting analysis of different human tissue extracts with specific antisera obtained against gastrin-binding proteins was performed. It was found that high molecular weight polypeptide zones of 120 kDa and 35 kDa were characteristic of the brain only. Autoantisera of patients with type A gastric disease reacted with some gastrin/cholecystokinin-binding proteins in human brain and mucosa including human brain polypeptide of 120 kDa. It is supposed that there are neurospecific gastrin-binding proteins (possibly gastrin/cholecystokinin receptors in the brain).     

Non-apoptotic Functions of Caspase-3 in Nervous Tissue

Some enzymes that have been recognized as "apoptotic" so far may be involved in important cellular processes not necessarily related to cell death in nervous tissue. The activity of caspase-3, an "apoptotic" enzyme, can be measured in normally functioning neurons. The results reported by several groups point to the possibility that caspases may be involved in nervous tissue function as top enzymes in the regulatory proteolytic cascade. A concept on a new mechanism of synaptic plasticity modulation involving caspase-3 has been formulated postulating a specific role of caspase-3 in normal brain functioning. The idea of synaptic plasticity modulation by caspase-3 is in line with data reported recently. For example, caspase-3 is possibly involved in the long-term potentiation (LTP) phenomenon since proteins that are key players of molecular mechanisms of LTP induction and maintenance are caspase-3 substrates. Experimental results on blocking LTP by a caspase-3 inhibitor confirm this concept.     

Identification of a putative isoform of the Na,K-ATPase beta subunit. Primary structure and tissue-specific expression

We have isolated cDNA clones from rat brain and human liver encoding a putative isoform of the Na,K-ATPase beta subunit. The rat brain cDNA contains an open reading frame of 870 nucleotides coding for a protein of 290 amino acids with a calculated molecular weight of 33,412. The corresponding amino acid sequence shows 98% identity with its human liver counterpart. The proteins encoded by the rat and human cDNAs exhibit a high degree of primary sequence and secondary structure similarity with the rat Na,K-ATPase beta subunit. We have therefore termed the polypeptides these cDNAs encode a beta 2 subunit with the previously characterized rat cDNA encoding a beta 1 subunit. Analysis of rat tissue RNA reveals that the beta 2 subunit gene encodes a 3.4-kilobase mRNA which is expressed in a tissue specific fashion distinct from that of rat beta 1 subunit mRNA. Cell lines derived from the rat central nervous system shown to lack beta 1 subunit mRNA sequences were found to express beta 2 subunit mRNA. These results suggest that different members of the Na,K-ATPase beta subunit family may have specialized functions.     

Comparison of acetylcholine and α-bungarotoxin binding sites in insects and vertebrates

1. The nervous tissue of locusts contains high affinity as well as low affinity binding sites for acetylcholine which display a similar nicotinic pharmacology.2. Hill plot analysis indicated a non-cooperative binding of acetylcholine.3. In membrane preparations from locust ganglia and mouse brain the number of binding sites for ACh was about ten fold lower than for BGTX, whereas in membranes from electric tissue both sites occurred in similar concentrations.4. Drug binding studies suggest that the high affinity binding sites for ACh and BGTX in preparations from insect and mouse are different; whereas in electric tissue both sites are very similar.5. Precipitation experiments using immobilized BGTX and specific antibodies indicated that in insect nervous tissue as in electric tissue the ACh and BGTX binding sites are located on the same receptor molecule and occupy distinct partially overlapping binding sites, whereas in the vertebrate brain both sites are located on distinct binding proteins.     

Are astroglial cells involved in morphine tolerance?

Morphine gives rise to a cascade of events in the nervous system affecting, among others, neurotransmitter metabolism. Tolerance develops for various effects shortly after administration of the drug. Also, physical dependence develops and can be demonstrated by precipitation of withdrawal reactions. Biochemical events in nervous tissue have been extensively studied during morphine treatment. This overview will focus upon brain protein metabolism since macromolecular events might be of importance for development of long-term effects, such as tolerance and physical dependence. Both dose-and time-dependent changes in brain protein synthesis and the syntheses of specific proteins have been demonstrated after morphine treatment, although methodological considerations are important. Different experimental models (animal and tissue culture models) are presented. It might be interesting to note that astroglial protein synthesis and the secretion of proteins to the extracellular medium are both changed after morphine treatment, these having been evaluated in astroglial enriched primary cultures and in brain tissue slices. The possibility is suggested that proteins released from astroglial cells participate in the communication with other cells, including via synaptic regions, and that such communication might be of significance in modifying the synaptic membranes during morphine intoxication.     

Immunohistochemical application of monoclonal antibodies against myelin basic protein and neurofilament triple protein subunits: advantages over antisera and technical limitations

Four monoclonal antibodies against guinea pig myelin basic protein (MBP), and four against subunits of bovine neurofilament triplet proteins (NF) were produced and their activity determined by enzyme-linked immunosorbant assay. The specificity and cross-reactivity of these eight monoclonal antibodies and one heterologous antiserum against each of the two central nervous system (CNS) antigens were examined in a histological study using the immunoperoxidase, antibody sandwich technique in rat and human brain tissue. Tissue sections were prepared from paraffin-embedded or fresh brain tissue that had been fixed with one of five different fixatives. The resulting immunoperoxidase labeling was then graded for intensity and examined for artifacts. One monoclonal antibody against MBP and one against NF resulted in labeling that was superior to that given by each of the antisera against their respective antigens. Of the five fixatives tested, a mercuric chloride-formalin solution gave the best preservation of these two antigens in rat and human brain tissue. The mercuric chloride-formalin solution was found to be superior to the other fixatives when immersion fixation was used, and was especially optimal when brains were perfused fixed. Three artifacts were encountered among the various antibody-fixative combinations that produced erroneous, but seemingly specific staining of Purkinje cells, neurons and axons, or astrocytes.     

Microheterogeneity of tubulin proteins in neuronal and glial cells from the mouse brain in culture

The microheterogeneity of the alpha and beta isoforms of tubulin in brain cells in culture was studied. The cells were prepared from two precise regions of the embryonic mouse brain (ED15), the striatum and the mesencephalon. It was possible to maintain virtually pure cultures of neuronal or glial cells up to 1 and 4 weeks in vitro, respectively. The tubulin heterogeneity of striatal and mesencephalic neurons was found to be very similar after a few days in culture. More precise examination of pure neurons from the striatum revealed that their tubulin content after 7 days in vitro exhibited the same degree of complexity as a control extract from a 4 day-old mouse brain. In fact, we could detect the presence of at least six alpha and nine beta tubulin isoforms. Among these isoforms a specific family of beta proteins (beta' tubulin) and the more acidic alpha proteins were present. Since these isoforms have, up to now, been found only in tubulin extracts prepared from the nervous system, our experiments suggest that they belong to the neuronal subpopulation of this tissue. This point is reinforced by their complete absence from the tubulin proteins extracted from pure glial cells even after several weeks in vitro. These results lead us to propose that brain tubulin microheterogeneity is associated with the presence of neurons and not of glia and may, therefore, play a specific role in maintaining neuronal shape and function.     

Evidence for specific selenium target tissues and new biologically important selenoproteins

After in-vivo labeling with [75Se]selenite the Se-containing proteins present in rat tissues were investigated by means of SDS-polyacrylamide gel electrophoresis. Thirteen Se-containing proteins or protein subunits with relative molecular weights of 12,100, 15,600, 18,000, 19,700, 22,200, 23,700, 27,800, 33,300, 55,500, 59,900, 64,900, 70,100 and 75,400 were detected in the tissue homogenates. The protein with the molecular weight of 23,700 was the subunit of glutathione peroxidase, which is the only selenoprotein so far known to have biological functions in animals. Most of these proteins were found in all tissues investigated but one was only detected in the testes and the spermatozoa and one was present mainly in the thyroid. With inadequate selenium intake there was a priority supply of the element to the brain, the reproductive and the endocrine organs, and at a molecular level to Se-containing proteins other than glutathione peroxidase. The results suggest important biological functions of these selenoproteins, especially in the specific target tissues.     

Tissue-specific N-glycosylation of the ClC-3 chloride channel

A commercially available polyclonal antibody against a rClC-3/GST fusion protein was used in order to investigate the tissue distribution of the ClC-3 chloride channel protein. The antibody appeared to be specific to rClC-3 since no cross-reaction could be observed with rClC-4 or rClC-5 proteins when overexpressed in Xenopus oocytes. In mouse, mClC-3 was preferentially expressed in the central nervous system, intestine, and kidney. To a lower extent, mClC-3 protein was also detected in liver, lung, skeletal muscle, and heart. Surprisingly, the electrophoretic mobility of mClC-3 differed in the various tissues. After enzymatic digestion of N-linked oligosaccharide residues of membrane proteins from brain, intestine, and kidney, mClC-3 was found to migrate at its calculated molecular mass. This study provides important information regarding the specificity of the used antibody, indicates that ClC-3 is widely expressed in mouse, and that mClC-3 undergoes differential tissue-specific N-glycosylation.     

Experimental approaches to the study of permeability of the blood-brain barrier for native autogenous proteins in chicken ontogenesis

The composition and distribution of serum proteins in blood, brain vesicles liquid, and nervous tissue has been studied in chicken embryos at 4-20 day of incubation using electrophoresis, immunoelectrophoresis, and fluorescent antibodies. At the 4-5 day of incubation, a difference in the protein composition of brain vesicles liquid and blood serum is apparent. This indicates that the border membrane of brain vesicles plays a part of the barrier. The vessels, preventing the transport of serum proteins to the nervous tissue, which are characteristic of hematoencephalic barrier of adult animals, develop in the embryo brain at the end of incubation period.     

Phylogenetic Survey of Proteins Related to Synapsin I and Biochemical Analysis of Four Such Proteins from Fish Brain

A phylogenetic survey of proteins immunologically related to Synapsin I, a major synaptic vesicle-associated phosphoprotein in mammals was carried out. Proteins antigenically related to Synapsin I were found by use of radioimmunoassay and other radioimmunochemical techniques in the nervous systems of several vertebrate and invertebrate species, which included birds, reptiles, amphibians, fish, echinoderms, arthropods, and mollusks. Four proteins present in fish brain, antigenically related to Synapsin I, were further studied and found to resemble mammalian Synapsin I in several respects. Like Synapsin I, the fish proteins were present in high amounts in nervous tissue, were enriched in synaptosomal fractions of brain where they were substrates for endogenous protein kinases, were acid extractable, and were sensitive to digestion by collagenase. In addition, two-dimensional peptide-mapping analysis revealed some homology between major phosphopeptide fragments of Synapsin I and the fish proteins. The results indicate that proteins related to Synapsin I are wide-spread in the animal kingdom.     

Pituitary adenylate cyclase activating polypeptide (PACAP): discovery and current status of research.

For last 2 years since PACAP was first discovered, many important findings on PACAP have been reported. cDNAs encoding the precursor proteins of PACAP in sheep, human and rat were cloned, and the precursor proteins characterized. PACAP was found in a high concentration in the central nervous system, adrenal medulla and testis. Immunohistochemical study indicated that PACAP containing neural fibers are present throughout the brain, including both internal and external zones of the median eminence. In the hypothalamus many PACAP positive cell bodies were demonstrated in the supraoptic nucleus and the paraventricular nucleus in various species. Four types of high affinity PACAP receptor were demonstrated. PACAP receptors in the central nervous system, pituitary, adrenal medulla and germ cells of the testis are highly specific for PACAP, and not shared with VIP. The PACAP receptor was solubilized and cross-linking of 125I-PACAP27 with the binding protein suggest that the molecular weight of the receptor is around 57,000. Various biological actions of PACAP were reported, but the physiological cellular events linked with PACAP-induced activation of adenylate cyclase remain to be investigated.